REPORT: COST-STSM-TD1002-181112-024118
Evaluation of nanomechanical properties of cells and soft gels using Atomic
Force Microscopy
Georgios PA Michanetzis1, Tomas Luque2 and Daniel Navajas2
1 STSM Applicant; Laboratory of Biomechanics and Biomedical Engineering,
University of Patras, Greece
2 Host; University of Barcelona, Spain
Period: 2012-11-18 to 2012-11-24
Abstract
Atomic force microscopy (AFM) has been used over the last fifteen years in cell
biology, not only as a microscopy method acquiring high resolution images, but also
as a useful “nanoindentation” tool for the evaluation of the viscoelastic properties of
living cells. However, many times the results of these studies are quite doubtful as
researchers often use different and not well characterised methodologies and do not
provide adequate information about data acquisition, processing and evaluation for
the different AFM systems used.
The scope of this COST-STSM was the evaluation of surface nanomechanical
properties of cells and soft gels using AFM, emphasizing on the methodology and the
problems of the technique. As a first outcome of this work the urgent need for
standardisation of the methods used was recorded.
Introduction
For more than fifteen years Atomic force microscopy (AFM) has been proved a useful
tool for imaging in the case of cell biology, as well as in other sciences, when high
resolution images are required. However, the possibility to use AFM also for the
quantification of normal and lateral forces exerted on the AFM tip, while interacting
with the surface of materials, has turned the AFM to a very useful nanoindentation
tool to evaluate surface nanomechanical properties of very soft materials, where
classic nanoindentation tips (fe Berkovich) fail to provide meaningful results.
AFM measurements have been used in numerous - more specific (1-7) or in a review
form (8,9) - studies, to evaluate cell mechanics in the case of normal, diseased, aged
cells etc aiming in a better understanding of cell mechanotransduction and of course,
eventually, in the establishment of novel diagnostic techniques. However, the main
problem of these studies is that they very rarely explain in details the methodology
followed, assumptions and limitations in processing and evaluating the data and this,
in many cases, make their results difficult to compare with results of similar studies
where fe a different AFM system is used or a different software is used for the
evaluation of the results. The problem appeared from the very beginning of this type
of research (10) and is still present after one and a half decade of studies (11).
Our aim was to perform a sample study using human neutrophils as well as soft gels,
as a reference material, to try (together with other studies) to provide standardization
instructions for suggested methodology and problem solution.
Materials and Methods
Two AFM systems were used in this study: One home-made system (University of
Barcelona, Spain) (Fig. 1) and a Multimode AFM system (Veeco, USA). For the
experiments we used MLCT cantilevers with 4 triangular tips with different spring
constants.
Figure 1: Home-made AFM system
Neutrophil polymorphonuclear granulocytes (PMNs) were isolated from blood using
a standard density gradient separation method.
Soft polyamide gels of different stiffness were also prepared from acrylamide /
bisacrylamide solutions and were used as reference materials.
Calibration of photodiode sensitivity
After initial laser beam alignment settings the deflection of the cantilever is monitored
as a function of sample/piezo position while the tip comes in contact with the sample
surface (usually a hard surface like glass). After the analysis of the data curve the
measured cantilever deflection z can be converted to the corresponding force F if the
spring constant of the lever k is known.
Figure 2: Determination of sensitivity and curve fitting (2)
Attention must be paid to the fact that depending on the reproducibility of the curves
obtained one should repeat the procedure several times to gain accuracy. Also curve
fitting and especially the correct choice of “zero” contact point may play an important
role. There is an apparent to provide standardization rules to solve these problems,
however this needs additional work and communication between several working
groups.
Accurate determination of spring constant
The accurate determination of the spring constant is also essential in order to correctly
evaluate force measurements. The cantilever’s bending stiffness or spring constant, k,
is usually estimated by its manufacturer, but this estimate can be quite gross, and
sometimes the error can exceed even 100%. For this reason, several methods have
been developed to arrive at a more accurate estimate. In our case k was determined
using the thermal tune method where the voltage noise recorded is related to the
thermal fluctuations of the cantilever position and angle.
Figure 3: Frequency vs. amplitude plot from a thermal tune (taken in air).
A) full-scale spectrum. B) zoomed thermal tab and fitted (blue line).
(figure taken from Asylum MFP 3D procedural operation manualette)
Unfortunately the result proved to be instrument-dependant for the two different types
of AFM systems used, which means this is also one field subjected to the need of
standardization rules.
Results and discussion
Results
Evaluation of the elastic modulus after recording the load-depth curve is also an
interesting and sometimes questionable procedure, as quite a few models exist and in
many cases of commercially available systems the built in software provides an
output value without any further information.
In our case the elastic modulus (E) of neutrophils was in the range of a few kPas and
was determined analyzing the data curve using a home-made software. The cell
experiments were performed just a few hours after venipuncture, as time plays an
important role in the evaluation of cell mechanical properties. In this case also, there
is a need to provide rules for several parameters as fe the buffers and solutions used,
fixing or labeling procedures that may be used in the case that AFM is combined with
other microscopic techniques (as epifluorescent / confocal microscopy or scanning
electron microscopy (SEM)), as well as for the suggested time of measurement, given
that cell activation may influence cell surface mechanical properties.
On the other hand the evaluation of the gels measured yielded elastic moduli between
10 and 500 kPa. In this case the procedure was much more standardized and is
suggested to be used as reference to compare measurements in the case of different
labs using different instrumentation.
Discussion
Atomic Force Microscopy has been proven to be a valuable tool in the case of cell
biology, not only for cell imaging, but also for evaluating cell mechanical properties,
providing information on the structure and function of the cells as well as of the
underlying cytoskeleton and cell organelles.
However, as it was pointed also by the outcome of this STSM, there are also a lot of
parameters that can influence the results of this type of studies, making it really
difficult in many cases to compare results from different groups using different AFM
setups. For example tip selection and selection of mode (contact, non contact,
tapping) can greatly influence the quality of the images obtained.
In the case of force measurements we may overall state that in order to get reliable
results there is an urgent need to set standard methods and rules followed by the
different groups in the evaluation of cell nanomechanics. Sensitivity, spring constant,
data processing and evaluation of the elastic modulus are some of the important
parameters we have to find standard and generally accepted ways to determine.
In addition there is also a need to get a better insight in the built-in software of the
different AFM systems used (especially commercial ones) in order to be also able to
compare their results.
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